We have previously described
the synthesis of ABEA-BY630, a fluorescent derivative of the adenosine
agonist NECA (5'-N-ethylcarboxyamidoadenosine) and described its agonist
activity at Gi/o-coupled human A1 adenosine
receptors expressed in CHO cells (Briddon et al. 2003). The aim
of the present study was to determine whether the same ligand was able
to stimulate Gs-coupled A2 adenosine
receptors that are endogenously expressed in human prostatic stromal cells.

Human prostatic stromal cells were isolated from benign prostatic tissue
and cultured as previously described (Abdul-Hamid et al. 2001).
Cells were grown from one cell line and functional assays performed on
cells of passage numbers 4-8. Cells were grown in 24-well cluster dishes
and [3H]cAMP
accumulation measured as described previously (Cordeaux et al.
2000). Where stated, cells were pre-incubated with the antagonists; ZM241385,
XAC or SCH58261 (100nM), 15 minutes prior to incubation with agonist.

In human prostatic stromal cells, the agonist NECA stimulated [3H]cAMP
accumulation in a concentration-dependent manner (pEC50=
5.66 ± 0.04, mean ± s.e. mean, n=19). Pre-incubation of
cells with the non-selective antagonist XAC caused a rightward shift in
the concentration-response curve to NECA(pKB
= 7.85 ± 0.08, n=3). The weakly A2A-selective
antagonist ZM241385 produced a similar shift (pKB
= 7.72 ± 0.04, n=3). The highly selective A2A
antagonist SCH58261 (100nM), however, did not alter the NECA response
(pEC50s; 5.27 ± 0.05 and 5.59
± 0.17, with and without SCH58261, respectively, n=3). Moreover,
the A2A-selective agonist CGS21680 only
produced a small [3H]cAMP
response (10 ± 4% of NECA maximum at 0.1mM) in these cells at a
concentration above 10µM (n=3). Hence, the effects of NECA in these
cells can be attributed to activation of the A2B
adenosine receptor.

The fluorescent ligand ABEA-BY630 also stimulated [3H]cAMP
accumulation in these cells with similar potency to that of NECA (pEC50
= 6.32 ± 0.08, n=4). This stimulation was completely inhibited
by preincubating the cells with XAC (1mM, 30 minutes, n=3). Relative to
NECA, however, ABEA-BY630 was a partial agonist (37 ± 5 % of NECA
maximum, n=4). This agonist may therefore be a useful tool for visualising
adenosine receptors in primary cells, and studying their function using
fluorescence-based techniques.